The Evidence for Contraceptive Options and HIV Outcomes (ECHO) trial found no substantial difference in HIV acquisition risk between women randomised to injectable depot medroxyprogesterone acetate (DMPA-IM), copper intrauterine device (IUD) or the levonorgestrel (LNG) implant. However, it remains unknown whether these contraceptives increase HIV risk relative to other forms of contraception or no contraception. This study investigated the impact of DMPA-IM, copper IUD and LNG implant on cervicovaginal inflammatory profiles previously associated with HIV acquisition, among a sub-cohort of ECHO participants. This study included 167 ECHO participants at the Setshaba Research Centre in Pretoria and MatCH Research Unit in Durban, South Africa. Eleven cytokines and antimicrobial peptides were measured in lateral vaginal wall swabs in duplicate using Luminex. Differences in baseline cytokine profiles were assessed using demographic data, including site, age, body mass index (BMI) and sexually transmitted infection (STI) status. Changes in cytokine concentrations were assessed using Wilcoxon signed ank test. Fold changes in cytokine concentrations were compared between arms using Mann-Whitney U test. P-values were adjusted for multiple comparisons using a false discovery rate procedure. Overall cytokine profiles were compared using principal components analysis and unsupervised hierarchical clustering. Mixed effects linear regression was used for longitudinal analysis and multinomial logistic regression was used to adjust for potential confounders that were assessed at baseline. Concentrations of IL-6 and MIP-3α were significantly higher in women enrolled at the Setshaba Research Centre compared to the MatCH Research Unit. Several immune mediators were elevated in younger women and this trend was significant for the pro-inflammatory IL1β and the chemokines IL-8 and MIP-1α. Women that were seropositive for herpes simplex virus type 2 (HSV-2) had significantly lowerconcentrations of MIP-1α. The copper IUD and LNG implant were associated with rapid increases in inflammatory markers following contraceptive initiation. Pro-inflammatory IL-1β and IL-6 and chemotactic IL-8, IP-10, MIP1⍺ and MIP-1β were significantly elevated one month following copper IUD insertion. No changes were evident at one-month post LNG implant insertion, however at three months, TNF-⍺, IP-10, MIP-3⍺ and SLPI were significantly raised relative to baseline. No significant changes in immune mediator concentrations were detected following DMPA-IM initiation but the trend was towards a decrease, particularly for SLPI. After adjusting for potential confounders, including site, age and infection status with chlamydia, gonorrhoea and HSV-2, IL-6 and IP-10 were significantly elevated in the copper IUD compared to the DMPA-IM arm at months 1 and 3, while IP-10 and SLPI were higher in the LNG implant arm at month 3 compared to the DMPA-IM arm. The copper IUD and the LNG implant are associated with increased cervicovaginal inflammatory markers that have been linked to HIV infection risk and the chemokine IP-10 appears to play a central role. Recent studies have demonstrated the importance of the interplay between inflammation, the microbiome, contraception and HIV risk. Continued research to understand these effects are critical for safe contraceptive use and to inform novel contraceptive development.

Every year thousands of cases of neurological disease go undiagnosed largely due to the vast number of potential causes, especially neglected are those thought to be of viral origin. The purpose of this study was to investigate the potentially novel causes of undiagnosed neurological disease in horses. An arbitrarily primed PCR was developed which allowed for the identification of unknown agents from cell culture and as well as tissue specimens. Shuni virus (SHUV) was identified in a cell culture isolate from a horse that had displayed severe neurological signs. This little known orthobunyavirus, had not been well studied since its discovery in the 1960’s and thus the focus became to further elucidate the role SHUV may play in neurological disease in South Africa. Two SHUV specific assays were developed and employed in a five year epidemiologic study. 497 horses and 143 other animals submitted to our surveillance program with febrile and neurological disease were screened for the presence of SHUV. 13 SHUV cases were identified, nine in horses and four in wildlife species. In horses symptoms ranged from mild febrile illness to severe neurological disease, where 45% of animals either died or were euthanized on humane grounds. All wildlife cases presented with paralysis, all cases proved fatal. A genome was amplified and characterised and SHUV’s (SAE 18/09) relationship to the prototype SHUV isolate and the Simbu serogroup fully clarified. Of significance was the finding that the prototype isolate’s sequence differed from SAE 18/09 at one of the M segment cleavage sites, such changes are known to affect pathogenicity. Finally due to the zoonotic potential of SHUV, a serological survey was conducted on veterinarians, to determine whether they may be at increased level of exposure due to occupational risk. WNV was used in comparison as zoonotic transmission of this virus had been documented and multiple studies conducted to analyse its sero-prevalance. 12.5% of veterinarians were found to have neutralizing antibodies to WNV and 4% to SHUV, these values correlate with what is seen in equine studies (WNV 8.7% - SHUV 1.9%), highlighting the zoonotic potential of these pathogens. / Thesis (PhD)--University of Pretoria, 2014. / Medical Virology / PhD / Unrestricted

Despite huge strides being made towards decreasing the number of individuals getting newly infected with HIV-1, and in reducing AIDS-related deaths, unfortunately current predictions are that the 2020 UNAIDS goals (90-90-90 targets, where 90% of people living which HIV-1 are diagnosed as such, from which 90% will will receive sustained antiretroviral therapy, resulting in viral suppression in 90% of these individuals by 2020) are out of reach. This of course means that the numbers of newly infected indivuals and AIDS-related deaths will be above the target derived from the 2020 UNAIDS goals. The development of an effective HIV vaccine could therefore be an important step towards realising these objectives. In work done for this thesis, a heterologous HIV-1 vaccine platform regimen was developed using antigen sequences from the predominant circulating HIV-1 subtype (subtype C) in South Africa. Specifically, this involved use of the envelope glycoprotein sequence of the CAP256 superinfecting virus (CAP256_SU) from the CAPRISA 002 cohort, and a mosaic Gag sequence which resulted in robust autologous Tier 2 neutralisation of CAP256_SU pseudovirions. The envelope glycoprotein sequence was modified so as to replace the native leader sequence with the tissue plasminogen activator leader, the furin cleavage site with a glycine rich flexible linker, and to introduce an I559P mutation. DNA and modified vaccinia virus Ankara (MVA) vaccines were generated where Env was truncated to gp150, thereby retaining the transmembrane domain and a partial cytoplasmic tail (Env). The Env sequence for the protein vaccine was further trimmed by removal of the transmembrane domain to give gp140, leading to a soluble, secreted protein (soluble Env). This allowed for the latter vaccine to be affinity purified using lectin (soluble Env (GNL)), and after generating stable cell lines, soluble Env yields were high enough to enable size exclusion chromatography which allowed isolation of the trimeric fraction of Env as determined by molecular weight (soluble trimeric Env). A Cterminal His-tagged version of soluble Env was generated as well. Surprisingly, the folding of Env-His was inferior to soluble Env, with a switch in profile from mainly trimeric Env to mainly monomeric Env. Nevertheless, soluble Env-His (GNL) and soluble trimeric Env-His were assessed for the presence of Env broadly neutralising antibody (bnAb) epitopes in an ELISA assay. The V3-glycan supersite (binding of bnAbs PGT128 and PGT135), the CD4-binding site (VRC01) and the V2-glycan site (PG9) were detected for both Env-His (GNL) and soluble trimeric proteins, whereas low signals for PG16, PGT145 and CAP256-VRC26.08, bnAbs which specifically recognise Env trimers in a native-like conformation, were only detectable for soluble trimeric Env-His. Soluble Env (GNL) was subsequently used as a protein vaccine in rabbits to test the immunomodulatory effects of the two adjuvants AlhydroGel (similar to alum) or the MF59-like squalene-based oil-in-water nano-emulsion AddaVax. Soluble Env (GNL) adjuvanted in AlhydroGel resulted in improved immune response in rabbits, with significantly higher serum binding antibodies to soluble Env (GNL) and scaffolded CAP256 V1V2-loop in comparison to AddaVax and unadjuvanted protein. Furthermore, significantly higher neutralisation titres to Tier 1A subtype C virus (MW965.26), in combination with an improved breadth to subtype C Tier 1A and 1B viruses, were observed in the AlhydroGel group. However, no neutralisation of Tier 2 viruses was detected. Nonetheless, AlhydroGel was selected as the best protein adjuvant for all further rabbit immunogenicity studies. Furthermore, in all subsequent experiments, soluble trimeric Env was used as a protein vaccine. DNA and recombinant MVA vaccines were generated using a membrane anchored gp150 (Env) with the aim that co-expression with mosaic Gag (GagM) would lead to the incorporation of Env into Gag virus-like particles (VLPs). Electron microscopy of cells expressing Env+GagM from DNA and recombinant MVA vaccines verified VLP formation from these constructs, and the presence of Env was observed in VLPs purified using a two-step OptiPrep gradient centrifugation protocol. The presence of Env bnAb epitopes in cellular membrane-bound Env was verified by qualitative immunofluorescent microscopy of live-cell stainings and a quantitative FACS assay. The same bnAb epitopes as for the Env protein vaccine were detectable, including bnAbs recognising only native-like Env trimers (PG16, PGT145 and CAP256-VRC26_08). However, expression levels of native-like Env trimers were lower, at approximately 20% when normalised to VRC01. These HIV-1 DNA, rMVA and soluble trimeric Env protein vaccines were tested in different heterologous vaccine platform immunogenicity studies in rabbits. These consisted of either priming with two recombinant MVA vaccines and boosting with three protein vaccines (MMPPP), or priming with DNA vaccines followed by two MVA vaccines, followed by two protein vaccines (DDMMPP). Furthermore, the inclusion of GagM into the DNA and MVA vaccines was compared to use of Env alone. Both vaccine regimens resulted in binding antibodies to soluble trimeric Env and a scaffolded CAP256 V1V2-loop; however, these were induced by MVA and protein vaccines, but not by DNA vaccines. Despite the lack of Env binding antibodies after DNA vaccination, better neutralisation was observed for the DDMMPP regimen compared to MMPPP, resulting in higher sera neutralisation titres towards vaccinematched, autologous Tier 2 CAP256_SU virus. Most encouragingly, when compared to Env alone, the inclusion of Gag (Env+GagM) into DNA and MVA vaccines improved the immunogenicity of the DDMMPP regimen even further. For Env+GagM DDMMPP, more animals developed Tier 2 neutralising antibodies, and improved titres, whereas Tier 2 neutralisation in general started to develop after fewer vaccinations, as for most rabbits this was observed after the second MVA inoculation. In an attempt to improve the spike density of Env on VLPs and the plasma membrane, two Env chimaeras were made replacing parts of gp41 with the corresponding elements of influenza A H5 haemagglutinin (HA2) (Env:HA2 chimaeras). Increased Env spike density was observed in a previous study using this strategy for the gp41 transmembrane domain and cytoplasmic tail (gp140HA2tr). A similar construct was generated here for CAP256_SU and a second chimaera was included replacing the whole of gp41 with HA2 (gp120HA2). Surprisingly, in experiments where VLPs were purified from OptiPrep gradients or the whole-cell bnAb FACS assay conducted with these Env:HA2 chimaeras, there was no evidence of increased spike density on VLPs or the plasma membrane as compared to Env. Furthermore, the folding of Env was severely impacted, especially regarding gp120HA2 where no binding of PG16, PGT145 and CAP256 VRC26.08 - bnAbs recognising native-like Env trimers - was observed. Although results for gp140HA2tr was improved over gp120HA2, in general the data for gp150 (Env) was superior in both the bnAb live-cell staining and FACS assay. Consequently, when both Env:HA2 chimaeras in combination with GagM were tested in the DDMMPP regimen, no improvement was observed with regard to autologous Tier 2 neutralisation. For rabbits receiving gp120HA2, no animals developed Tier 2 nAbs, whereas for gp140HA2tr, Tier 2 neutralisation in general developed later and to lower titres compared to Env+GagM. In conclusion, different HIV-1 DNA, recombinant MVA and protein vaccines were generated and characterised both in vitro and in vivo, leading to a vaccination regimen that induced both high titre Env binding and vaccine-matched Tier 2 neutralising antibodies in rabbits. Furthermore, a new Env sequence, the first from the South African CAPRISA cohort, has been added to the small list of Env sequences that can induce Tier 2 neutralisation.

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Includes bibliographical references (leaves 191-221).

Includes bibliographical references (leaves 133-147) / In transplantation, vascularized organs often suffer the consequences of ischemic damage as well as reperfusion injury following the reestablishment of blood flow. The induced ischemialreperfusion (I/R) damage is usually associated with the accumulation of injurious complement components. The vaccinia virus complement control protein (VCP) has the ability to simultaneously inhibit the classical and the alternative complement pathways by binding to the early components C3b and C4b. The complement component C3 is known to be the central route to all of the known complement activation pathways. As a result, it is involved in a number of complement-mediated ailments including renal ischemia/reperfusion injury. The objectives of this study were to initially evaluate the in vitro roles of the natural VCP and the humanized recombinant VCP (hrVCP), and then to establish their in vivo roles in a renal I/R injury model.

Includes bibliographical references (leaves 161-184). / This study shows that HIV-specific cervical T cells can be isolated by cytobrushing and in vitro polyclonal expansion is a useful approach to increase the number of T cells available from mucosal sites. Dynal beads (1:1) in the presence of IL-2, IL-7 and IL-15 resulted in the best yields of cervical T cells while anti-CD3 in the presence of IL-2 best conserved the ex vivo T cell profile. Expanded T cell lines, irrespective of expansion method used, generally maintain their cytokine response profile to HIV anti- gens. This study shows that HIV Gag-specific blood and cervical T cells were largely mono-functional with polyfunctional T cells being detected in women with high blood CD4 count and low plasma viral load. This study confirms that HIV-specific Gag T cell responses detected in the polyclonal expanded female genital tract T cells are associated with those measured in blood during HIV infection.

The development of an effective HIV-1 vaccine remains a global priority. Neutralizing antibodies (nAbs), which block infection by cell-free virus, are likely to be an important response for vaccines to elicit. However, evidence from the RV144 vaccine trial and non-human primate vaccine studies suggest antibody-dependent cellular cytotoxicity (ADCC) responses, which target virus-infected cells, may also be protective. This thesis uses deep sequencing, together with immune assays, to characterise HIV-1 Envelope evolution associated with both nAb and ADCC responses in early infection, and investigates broadly neutralizing and non-neutralizing monoclonal antibody ADCC activity against subtype C viruses. Recent advances in deep sequencing approaches, coupled with the primer ID method which barcodes each viral genome, enabled us to generate thousands of viral sequences to accurately track viral population dynamics in early infection. In all participants investigated, there was a significant drop in the relative frequency of wildtype (WT) virus following nAb responses. However, in three of the seven participants, when controlling for changes in viral load (VL) over time, we observed that the WT load (frequency of the WT residue x total VL) remained relatively stable despite an effective nAb response. Instead, there was an outgrowth of the escaped virus with a concomitant increase in viral loads. We found that nAbs were inefficient at blocking cell-cell transmission of early WT and escape viruses, identifying this as one mechanism by which viruses may persist despite the presence of nAbs. These results suggest that other antibody effector functions such as ADCC, which target infected cells, may be important to elicit in a protective HIV-1 vaccines. If ADCC responses are important in controlling viral populations, one would expect to find evidence of viral escape from these responses. In all nine participants investigated, we found ADCC responses emerged prior to nAb responses, and in three individuals we observed sequence changes prior to detectable nAbs. To evaluate if these changes were due to ADCC pressure on the virus, we introduced select mutations into infectious molecular clones encoding the cognate early/acute envelope (Env-IMCs). In one participant, the mutation introduced conferred resistance to both nAb and ADCC responses, while in two participants, mutations were identified which resulted in resistance to ADCC but had no effect on neutralization, suggesting escape from ADCC. Longitudinal analysis in one of these participants, which targeted the CD4- binding site, revealed three distinct escape pathways, of which two conferred resistance to ADCC, and confirmed that ADCC responses can directly drive viral evolution in vivo. Finally, we investigated the ADCC activity of eleven anti-HIV-1 monoclonal antibodies (mAbs), including seven broadly neutralizing antibodies (bnAbs) and four non-neutralizing antibodies (nnAbs), against a panel of nine acute subtype C Env-IMCs. We found bnAbs had low to moderate ADCC breadth (11-66%). In contrast, while the two V2 nnAbs we tested were narrow and weak, the two nnAbs targeting CD4-induced epitopes (A32 and C11) mediated the broadest (78-100%) and most potent (0.06-0.81 μg/mL) ADCC against this panel. In addition, a nonlinear relationship was found between ADCC activity and strength of mAb binding to the infected cell surface (rs = -0.5309, p=0.0001). In conclusion, in contrast to studies which evaluated limited number of sequences, utilizing deep sequencing approaches, we found that the WT load remained relatively stable following early nAb pressure, albeit at lower relative frequency to the escape variant. Evasion of antibody responses through cell-cell transmission may contribute to the persistence of WT virus, providing further motivation for the importance of antibody effector functions that target infected cells in a protective HIV-1 vaccine. For the first time, we provide evidence of ADCC-mediated immune pressure in early infection, showing that these responses can exert selective pressure on HIV-1. However, the limited number of sequence changes relative to those observed following nAb pressure suggests that this response does not put as much selective pressure on the virus as nAbs. Lastly, the moderate breadth of bnAb ADCC activity provides evidence that there are common epitopes on free virions and on the surface of infected cells. This indicates bnAbs with potent and broad ADCC should be identified to include in antibody-based treatment and cure strategies, which aim to eliminate infected cells. Altogether, these data suggest that while eliciting nAbs should be the primary goal of HIV-1 vaccine design, ADCC-mediating antibodies may also play an important role.